The Effect of miR-505-5p on Inhibition of Serum Uromodulin Ameliorates Myocardial Inflammation and Apoptosis Induced by Ischemia-Reperfusion

Abstract

Background: It has been found that miR-505-5p is closely related to cardiovascular metabolic risk factors. Nonetheless, there is little research analyzing miR-505-5p for its role as well as molecular mechanism in myocardial injury caused by ischemia-reperfusion (I/R).

Methods: This work utilized quantitative reverse transcriptase PCR (qRT-PCR) for detecting miR-505-5p and serum uromodulin (sUmod) levels. sUmod, interleukin-1beta (IL-1β), IL-6, IL-10, caspase7, caspase9, tumor necrosis factor-alpha (TNF-α), Bax, and Bcl-xL expression was detected by western blot. Bioinformatics database was used for target prediction and miR-505-5's target was determined by luciferase reporter gene assay.

Results: Relative to sham group, sUmod was highly expressed within myocardial I/R injury (MIRI), whereas sUmod silencing significantly decreased the heart weight/body weight ratio, reduced serum myocardial enzymes expression, ameliorated I/R-mediated myocardial apoptosis, and inflammation. TargetScan bioinformatics database and luciferase reporter genes confirmed that sUmod was miR-505-5p's direct target gene, besides, miR-505-5p overexpression significantly improved the myocardial injury score, increased IL-10, decreased TNF-α, IL-1β, IL-6 expression, decreased caspase7, caspase9, Bax expression, and increased Bcl-xL expression. More importantly, overexpression of sUmod abolished miR-505-5p overexpression's role in I/R-mediated myocardial apoptosis and inflammation.

Conclusion: miR-505-5p can improve I/R-mediated myocardial apoptosis and inflammation by targeting sUmod. In this study, miR-505-5p is related to MIRI pathogenesis, which provides the new possible targeted therapy in patients with MIRI.

Figure 1

sUmod was highly expressed in myocardial I/R injury. (a and b) According to the target prediction of bioinformatics database, sUmod was highly expressed in myocardial I/R injury. (c and d) Western blot analysis showed that sUmod was also highly expressed in myocardial I/R injury. (e) qRT-PCR was used to analyze the expression levels of sUmod mRNA in each group.(f)Heart weight/body weight ratio in each group. (g–j) The expression levels of serum myocardial enzymes BNP (g), LDH (h), cTnl (i), and CK (j). ^∗P < 0.05 vs. Sham; ^#P < 0.05 vs. I/R.

Figure 2

sUmod silencing ameliorates I/R-induced myocardial inflammation and apoptosis. (a and b) Pathological changes and injury score in myocardial I/R injury (HE staining, bar = 100 μm). (c–g) Western blot was used to quantitatively analyze the expression of IL-1β, IL-6, TNF-α, and IL-10 in myocardial I/R injury. (h–l) Western blot was used to quantitatively analyze the expression of caspase-7, caspase-9, Bax, and Bcl-xL in myocardial I/R injury. ^∗P < 0.05 vs. Sham; ^#P < 0.05 vs. I/R.

Figure 3

sUmod was a direct target gene of miR-505-5p. (a) The putative miR-505-5p-binding sequence in the 3′-UTR of sUmod mRNA. (b) Luciferase reporter assay of sUmod-WT and sUmod-Mut constructs in H9C2 cells transfected with ov-miR-NC or ov-miR-505-5p. (c) qRT-PCR were used to analyze the expression of miR-505-5p in H9C2 cells transfected with ov-miR-NC or ov-miR-505-5p. (d–f) Western blot and qRT-PCR were used to analyze the effects of ov-miR-505-5p and ov-sUmod on sUmod protein and mRNA expression.^∗P < 0.05 vs. ov-miR-NC; ^#P < 0.05 vs. ov-NC; ^&P < 0.05 vs. ov-miR-505-5p and ov-sUmod.

Figure 4

miR-505-5p overexpression attenuated I/R-induced myocardial injury. (a) The expression of miR-505-5p in I/R myocardium with ov-miR-505-5p and ov-sUmod was analyzed by qRT-PCR.(b–d) Western blot and qRT-PCR were used to analyze the effects of ov-miR-505-5p and ov-sUmod on sUmod protein and mRNA expression in myocardial I/R injury. (e) The effect of ov-miR-505-5p and ov-sUmod on heart weight/body weight ratio. (f and i) The effects of ov-miR-505-5p and ov-sUmod on serum cardiac enzymes BNP (f), LDH (g), cTnl (h), and CK (i). ^∗P < 0.05 vs. Sham; ^#P < 0.05 vs. I/R; ^&P < 0.05 vs. ov- miR-505-5p.

Figure 5

miR-505-5p overexpression attenuated I/R-induced myocardial inflammation and apoptosis. (a and b) The Effects of ov-miR-505-5p and ov-sUmod on myocardial pathological changes and injury score after myocardial I/R injury (HE staining, bar = 100 μm). (c–g) Quantitative analysis of IL-1β, IL-6, TNF-α, and IL-10 in myocardial I/R injury with ov-miR-505-5p and ov-sUmod by western blot analysis. (h–l) Quantitative analysis of caspase 7, caspase 9, Bax, and Bcl-xL in myocardial I/R injury with ov-miR-505-5p and ov-sUmod by western blot analysis. ^∗P < 0.05 vs. Sham; ^#P < 0.05 vs. I/R; ^&P < 0.05 vs. ov-miR-505-5p.